Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic neurons in the ventral midbrain (VM), resulting in striatal dopamine (DA) depletion. The neuronal loss is severe for DA neurons in the substantia nigra (SN), while the neighboring ventral tegmental area (VTA) DA neurons are relatively spared. Dysregulation of neuronal DA homeostasis leading to a buildup of cytosolic DA and resulting oxyradical stress has been suggested to play an important role in PD pathogenesis, although this hypothesis does not explain the differential susceptibility of the subpopulations of ventral midbrain DAergic neurons. To study the difference in catecholamine homeostasis between different neuronal populations, we recently developed intracellular patch electrochemistry (IPE), a technique that directly measures cellular concentrations of cytosolic catecholamines. We have now adapted this approach for measuring cytosolic DA in cultured VM neurons. Preliminary data confirm the relationship between high levels of cytosolic DA and neurotoxicity and demonstrate that neuronal death depends on the dose (concentration * time) of elevated cytosolic DA. Importantly, SN neurons display significantly higher cytosolic DA concentrations than VTA neurons when treated with identical L-DOPA doses, which also correlated with greater susceptibility of SN neurons to L- DOPA-induced toxicity. This project will be focused at determining biochemical/cellular mechanisms responsible for the difference in the maintenance of cytosolic DA between SN and VTA neurons. In particular, our preliminary data indicate that regulation of basal Ca2+ concentration, which has recently been suggested to underlie higher vulnerability of SN neurons to various types of stress, controls cytosolic DA homeostasis, a process that we will study in detail. IPE will be used as a tool to screen for changes in cytosolic DA in neurons treated with a range of drugs;biochemical assays will be employed to confirm suspected protein-protein interactions. This study will be important for understanding the mechanisms leading to differential susceptibility of neuronal populations to stress and for the development of treatment strategies that might protect the remaining SN DAergic neurons in PD patients. PUBLIC HEALTH RELEVANCE: The project will try to resolve a major controversy in the neuropathology of Parkinson's disease (PD): why are neurons in the substantia nigra particularly vulnerable in PD, despite their higher resistance to stress in experiments in vitro? We hypothesize that the rate of cytosolic DA turnover is dependent on cellular Ca2+ concentration, and, specifically, on the presence of the L-type Cav1.3 channel, which is responsible for pacemaking activity in SN neurons. This study will be important for understanding the mechanisms leading to differential susceptibility of neuronal populations to stress and for designing therapeutic strategies for the treatment of patients with PD or those exposed to toxins or drugs that induce a degeneration of DAergic neurons. 1